The goal of this proposal is to evaluate lentiviral gene vectors expressing a novel T cell receptor (TCR), that binds directly to MUC1 on the surface of cancer cells, as a new therapeutic agent for the treatment of adult carcinoma. The TCR, the lentiviral vector used to induce its expression in transduced immune cells, and the target itself are unique with respect to the standard means by which adoptive immunotherapy is proposed for treating cancer. The MUC1-specific TCR identified by Dr. Olivera Finn recognizes a unique antigenic structure on hyopglycosylated MUC1 (found on 85% of all carcinomas but not on normal cells;including breast, prostate, head and neck, and lung cancer) as opposed to a peptide bound by MHC. This makes our TCR a truly universal reagent that is not dependent on MHC-restricted antigen presentation by the tumor. We will create a single chain "third-generation" vector that will express a chimeric antigen receptor (CAR) containing CD28, CD137 (4-1BB) and CD3-zeta chain signaling elements. These vectors will be evaluated in both T cells and NK cells. The vector system used will be Lentigen's proprietary backbone construct that includes the latest safety elements as well as additional selection markers such as the tmpk "suicide" gene. The target, hypoglycosylated MUC1, is well-established as a unique antigenic signature of cancer and is an ideal target for immune cells expressing a CAR. In this proposal we will test the central hypothesis that lentiviral engineered T cells with significant anti-tumor efficacy can be created by developing improved "third generation" chimeric antigen receptors (CAR) that will target and specifically kill MUC1+ cancers. These CAR-expressing cells will undergo the same mechanism of clonal expansion into effector and memory populations that occurs during conventional TCR-pepMHC interactions. Moreover we will also test a corollary hypothesis that NK cells transduced with CAR will also serve as potent anti-MUC1+ effector cells. Through our detailed analysis of the native TCR, first generation CAR, third generation CAR, and different ways of activating immune cells for lentiviral transduction and subsequent evaluation of effector function, we will create a definitive product, with a consistent means of implementing its use that is translatable to the clinical setting. We will evaluate both standard means of immune cell activation (IL-2 plus anti-CD3), newer generation methods (anti-CD3/anti- CD28 beads), and cutting edges techniques (cell based artificial antigen-presenting cells that also include CD137 signaling). We anticipate that the results generated in this proposal will serve as the basis for Phase II SBIR studies, during which we will test the ability of T cells or T cells +NK cells expressing CAR to control or eliminate tumor growth in a Phase I clinical trial. The lentiviral production capability of Lentigen Corp., combined with the translational MUC1 research by Dr. Olivera Finn at the University of Pittsburgh makes this a realistic first step in carrying out clinical trials. These trials will be the first to evaluate non-MHC restricted TCR activity that will benefit patients suffering from MUC1+ cancer. PUBLIC HEALTH RELEVANCE: The goal of this research proposal is to develop a new anti-cancer agent that will benefit patients for which we currently do not have effective therapy. This therapy is based on the activation of immune cells outside the body, giving activated immune cells a new receptor on their surface that recognizes cancer cells, and then introducing these cells back into the body. We anticipate these modified cells will eliminate cancer cells upon re-infusion and benefit patients failing other therapies. This therapy, if proved successful, will have a significant impact for cancer patients and healthcare providers designing therapy for them in the United States and worldwide.